Circ_HOMER1及其靶miRNAs调控人脐带间充质干细胞成骨分化的机制研究

Human umbilical cord mesenchymal stem cells (hUCMSCs) are promising seed cells in bone tissue engineering, but their capability of bone formation still needs to be improved. Clarify their differentiation mechanism can help to improve their osteogenesis capability and clinical application. Circular RNA(circRNA) plays an important role in regulation of cell growth and other biological behaviors via its target microRNAs(miRNAs)( Nat Commun, 2016). Our group has combined with circRNA microarray, miRNA microarray and gene expression microarray to screen out circ_HOMER1 and its target miRNAs. And upregulation of circ_HOMER1 could make hUCMSCs possess osteogenic phenotype. In order to validate the hypothesis that the circ_HOMER1 could regulate the osteogenic differentiation of hUCMSCs via its target miRNAs, this project is preparing to explore the effects and mechanism of circ_HOMER1 and its target miRNAs on osteogenic differentiation of hUCMSCs in vitro; to further validate this regulatory mechanism in vivo in nude rats. The results will not only clarify the osteogenic differentiation mechanism of hUCMSCs and enhance their capability of bone regeneration, but also build on a solid theoretical and applied foundation for differentiation regulation of stem cells by circRNA.

人脐带间充质干细胞（hUCMSCs）是骨组织工程极具优势的种子细胞，但其成骨效果仍待提高，深入阐明其分化调控机制，可为提高其成骨效果并推动临床应用提供新策略。最新研究表明circRNA可通过其靶miRNAs调控细胞生长等生物学行为（Nat Commun，2016）。课题组前期联合circRNA、miRNA及表达谱芯片筛选出circ_HOMER1及其靶miRNAs，并初步证实circ_HOMER1可促进hUCMSCs成骨表型。故推测circ_HOMER1可通过其靶miRNAs调控hUCMSCs成骨分化。为验证此假说，本项目拟通过体外实验研究circ_HOMER1及其靶miRNAs对hUCMSCs成骨分化的影响及其调控机制；用裸大鼠体内实验进一步验证这一调控作用。预期研究结果有助于进一步阐明hUCMSCs成骨分化机制，提升其骨修复效果，并为circRNA调控干细胞分化提供理论与实践依据。

本课题通过高通量转录组测序的生物信息学分析、细胞生物学的分子和表型实验以及动物体内研究，探究了circ-CTTN及其靶miRNA对hUCMSCs成骨分化的影响及调控机制。.前期对基因芯片结果进行分析，我们筛选出了差异表达的circ-HMOER1作为目标分子进行研究。通过对circ-HOMER1的研究发现，其基因序列较长，过表达载体转染效率低下不利于开展后期实验。故而进行高通量测序后，根据数据筛选出新的目标分子circ-3766（通用名为circ-CTTN），其靶基因中含有多种成骨分化相关的基因。使用腺病毒转染技术成功构建circ-CTTN过表达载体，对hUCMSCs进行转染后发现，成骨相关基因的表达水平显著高于对照组。茜素红及碱性磷酸酶染色也表明过表达circ-CTTN组的hUCMSCs矿化诱导后成骨表型更明显。为进一步确定预测的下游miRNA:miR-1471能否调节成骨分化，分别用miR-1471抑制剂及miR-1471的mimic转染hUCMSCs。同时进一步向成骨方向诱导7d分别检测成骨分化相关基因mRNA和蛋白水平的变化。结果提示：抑制miR-1471表达能够促进促成骨分化相关基因的表达及成骨表型。.最后，在SD大鼠颅骨临界骨缺损模型上，采用生物相容性良好的水凝胶支架材料GelMA分别负载转染过表达载体、空表达载体、无载体的circ-CTTN的hUCMSCs。分别行2、4、8周取样，通过Micro CT分析及组织切片染色检测骨再生效果。结果显示，circ-CTTN过表达组的颅顶周围骨小梁形成更多，骨小梁更致密。.综上结果：本研究初步证明了circ-CTTN是hUCMSCs成骨分化过程中的关键调控circ RNA。